EL.SEVIER FEMS Microbiology Letters 134 ( 1995) 63-67 Binding of hemoglobin by Porphyromonas gingivalis Atsuo Amano, Masae Kuboniwa, Kousuke Kataoka, Katsuko Tazaki, Eiji Inoshita, Hideki Nagata, Hiroo Tamagawa, Satoshi Shizukuishi * Department zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Prel,entir,e Dentist?, Osaka Unit,er.Gp Faculty of Dentistry. I-X Yamadaoka. Suita. Osaka 565 , Japan Received 22 September 1995: accepted 26 September 1995 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSR Abstract In this study, we investigated whether Porphyromonas gingicalis can bind hemoglobin as an initial step in the acquisition of heme from hemoglobin. The binding of human hemoglobin by P. gingicalis cells was determined using [ 3H]hemoglobin. Hemoglobin binding occurred rapidly, reversibly and specifically. A Scatchard analysis of the binding data generated a linear plot, indicating a single population of binding proteins. The apparent K, was 1.0 + 0.19 X IO-’ M and there were 3.2 5 0.76 X IO4 binding sites per cell. Hemoglobin binding was inhibited by unlabeled human hemoglobin but not by hemin and protoporphyrin IX. The binding was only partially inhibited by human serum albumin, transferrin, lactoferrin. catalase and cytochrome c. These results suggest that the ligand recognized by the binding protein may not be the heme moiety. The binding of hemoglobin considerably increased when the organisms were grown under hemin-limited conditions. Hemoglobin bound to outer membrane proteins extracted from P. gingivalis cells on a dot blot binding assay and binding ability was lost after heating bacterial proteins. These results suggest that P. gingicalis cells interact with human hemoglobin through specific binding sites on their surfaces as a preliminary step in iron acquisition. Kewvrdst Porphyromonas ,qin,qil,a/is; Hemoglobin: Binding 1. Introduction The availability of iron in gingival crevicular fluid may be significant for the growth and virulence of the periodontopathogen Porphyromonas gingiualis [l]. Lactoferrin, transferrin, and hemoglobin are known constituents of crevicular fluid and probably support the growth of periodontopathogens in vivo [2]. We reported that P. gingiualis can utilize hemoglobin much more efficiently than other iron sources such as transfetrin, hemin, and inorganic * Tel: + 8 I (6) 876-57 11: fax: + 8 1 (6) 876-793 1. iron compounds [3]. Although hemoglobin is imme- diately bound by haptoglobin in the host environ- ment, the proteolytic capability of P. gingil~alis may contribute to the degradation of this host hemo- globin-sequestering protein, and liberate hemoglobin for binding and uptake into cells [3,4]. Thus, it is likely that a major source of iron for the growth of P. gingicalis is hemoglobin in gingival crevicular fluid. It is not clear how P. gingiculis acquires iron from hemoglobin. Whole P. gingidis, purified outer membranes, and lipopolysaccharide non-specifically bind to hemoglobin and hemin [5-g]. These studies, however, did not define the specificity of this bind- 0378-1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDI 0.778.1097(95)00382-7 Downloaded from https://academic.oup.com/femsle/article-abstract/134/1/63/524437 by guest on 05 June 2020